Gold nanoparticles (GNPs) act as less toxic and effective drug carriers with many specific properties that are particularly useful for biomedical and pharmaceutical applications. The present study was conducted to determine the anti-tumor properties GNPs. The new preparation technique is quick and cost-effective. GNPs formation was confirmed using various characterization techniques including X‐Ray diffraction (XRD) analysis, field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectrometer (EDX), and transmission electron microscopy (TEM). XRD, FES-EM, and TEM experiments have confirmed the formation of GNPs which shows that the prepared particles are spherical inform and have a smooth surface with an average size of 24.32 ± 1.32 nm. Multiparametric analysis of GNPs toxicity at the level of individual cells using flow cytometry and cellular imaging-based approaches such as High Content Screening (HCS) have played key roles in the detection of toxicity and classification of compounds based on observed patterns of reversible and irreversible cellular injury. The effect of GNPs on the MCF-7 cell line regarding cell viability, cell nucleus morphology, membrane permeability, potential mitochondrial permeability, and cytochrome C release was further confirmed using a High Content Screening array scan via multi-parametric kit. Results indicated that the viable count of MCF-7 cells was highly decreased by treated with GNPs. Nuclei stained with Hoechst blue appeared to be mostly condensed in response to the treatment with the GNPs (1.5- fold increase in nuclear fluorescence intensity), with significant differences (p < 0.0263) as compared with untreated cells. Cell membrane permeability was significantly (p = 0.0019) upon GNPs showing an increase of 1.9-fold, as compared with the untreated MCF-7 cells. GNPs induce a significant (p = 0.0053) decrease in mitochondrial membrane potential by 32.7%, in comparison to the untreated cancer cells with significant induction of the release of cytochrome C (p = 0.0016) was recorded after exposure to the GNPs (0.43-fold increased mean intensity), The outcomes of the present study propose the high feasibility for using GNPs as a novel anticancer drug.